*2.6. Analytical PCR*

To screen for target-gene disruption in drug-resistant transfectant cell lines, genomic DNA was isolated from non-clonal populations of *eGFP-*deletion mutants and analysed by PCR. Genomic DNA was isolated using ISOLATE II Genomic DNA Kit (Bioline, Luckenwalde, Germany).

To test for the presence of the *eGFP* ORF and integration of the drug-resistance genes (*BSD*, blasticidin-S deaminase; and *PAC*, puromycin N-acetyltransferase) in the *eGFP* mutants, 1 µL of isolated DNA was mixed with 1 × iProof high-fidelity PCR master mix (Bio-Rad), 0.4 µM each forward and reverse primers, and 12% DMSO in a 25.5 µL total volume. In parallel, a technical control PCR (to demonstrate the presence of DNA in the analysed samples) was performed by amplifying a fragment from the *L. donovani HSP23* or *L. braziliensis actin* ORFs. PCR steps were 3 min at 98 ◦C followed by 30 cycles of 30 s at 98 ◦C, 30 s at 60 ◦C, 30 s at 72 ◦C followed by a final elongation step for 5 min at 72 ◦C.

The *Leishmania* wild-type and parental cell lines were included as controls. 10 µL of each PCR reaction was run on a 1% agarose gel to check for the presence of the expected product. The list of primer pairs used is given in Table S1.
